Molecular Mechanism Of Rice Floral Transition Modulated By RID1 Recruiting Histone Modifiers

Heading date determines the geographical distribution of rice and is one of the key agronomic traits affecting yield.Rice a model for crop functional genome research.Research on the function of rice flowering genes and the molecular regulatory network has considerable theoretical and practical significance for the regulation of crop growth and improving the regional adaptability and yield of rice.Previous studies showed that Rice Indeterminate 1(RID1),the master switch for rice floral transition,encoded a C2H2 type zinc finger transcription factor.The mutation of RID1 influences the fundamental flowering pathways and prevents the expression of two vital florigen genes Hd3 a and RFT1,resulting in the failure of rice to complete the transition from vegetative to reproductive stage.Furthermore,both Hd3 a and RFT1 promoter regions contain the cis-element for RID1 binding,thus speculating that RID1 perhaps directly regulate the expression of Hd3 a and RFT1 to initiate rice floral transition.Through protein interaction studies,this study identified the interaction of RID1 with the gibberellin signal transduction repressor DELLA(SLR1 in rice)and histone modifiers SDG720,SDG722,and Os MED25.By generating mutants or overexpression materials of SLR1,SDG720,SDG722 and Os MED25,it was found that they all affected the process of rice floral transition.Further histone modification and chromatin status analysis showed that RID1 influenced the chromatin condensation status of Hd3 a and RFT1 promoter regions by recruiting histone modifiers SDG720,SDG722,and Os MED25.SLR1 competed with SDG722 and Os MED25 for binding to RID1,and the abundance of SLR1 gradually decreased during rice development.Hence,SLR1 might gradually eliminate the inhibition of RID1,enabling it to recruit histone modifiers to advance the expression of rice florigen genes(Hd3a and RFT1),thereby finely regulating rice floral transition.The chief outcomes are as follows:1.SLR1,a key regulator of the gibberellin pathway,affects rice floral transitionThe gibberellin synthesis mutant ga3ox-2 and SLR1 gain-of-function mutant SLR1-OX exhibited late-flowering phenotype under long-or short-day conditions,indicating that SLR1 negatively regulated rice floral transition.The detection of flowering genes' expression revealed that SLR1 delayed flowering primarily by inhibiting the expression of key floral promoting gene Ehd1 and florigen genes RFT1 and Hd3 a,indicating that SLR1 negatively regulated rice flowering through the Ehd1-RFT1/Hd3 a pathway.The m RNA and protein abundance of SLR1 in leaves gradually decreased with the development of rice,and the later the leaf was produced,the lower the expression of SLR1.In addition,overexpression of the C-terminal protein of SLR1 also delayed rice heading under longand short-day conditions,while overexpression of the N-terminal protein of SLR1 had no effect,indicating that only the C-terminus of SLR1 could delay rice heading date.2.SLR1 interacts with the C-terminus of RID1 and inhibits its transcriptional activityYeast two-hybrid,LCI,pull-down,Bi FC and Co-IP assays all confirmed that RID1 could directly interact with DELLA protein SLR1.SLR1 interacted with the C-terminus of RID1 and inhibited its transcriptional activity.These consequences indicated that SLR1 directly bound with the transcriptional regulatory domain of RID1 to inhibit its transcriptional activity.Ch IP experiments showed that the deposition of SLR1 at Hd3 a and RFT1 loci in rid1 decreased rather than disappeared completely,indicating that the deposition of SLR1 at Hd3 a and RFT1 loci partially relied on RID1.3.RID1 recruits histone modifiers Os MED25,SDG720 and SDG722Yeast two-hybrid results revealed that RID1 interacted with the mediator subunit Os MED25,and with the proteins SDG720 and SDG722 containing SET-domain.Pulldown and Co-IP assays further verified their interaction.These outcomes indicated that RID1 could recruit histone modifiers such as mediator and methyltransferase through protein interaction.Both Os MED25 and SDG722 interacted with the C-terminus of RID1 instead of its N-terminus,indicating that Os MED25 and SDG722 bound to the transcriptional regulatory domain of RID1 without affecting its DNA binding ability.4.Os MED25 positively regulates rice floweringOs MED25 was highly homologous to Arabidopsis MED25 and was widely expressed in various tissues.The loss-of-function mutant osmed25 generated by CRISPR/Cas9 displayed an obvious late-flowering phenotype under both SD and LD conditions,and resulted in significant suppression of the transcripts of Ehd1,RFT1,and Hd3 a.These outcomes suggested that Os MED25 positively advanced rice flowering and wasn't affected by photoperiod.Moreover,overexpression of Os MED25 failed in restoring the nonflowering phenotype of rid1,indicating that Os MED25 functioned upstream of RID1 to regulate rice flowering.5.Both SDG722 and SDG720 positively regulate rice floral transitionSDG722,encoding a SET domain protein,was the homologous protein of Arabidopsis ATXR2.The loss of SDG722 function caused a late-flowering phenotype of rice under both SD and LD conditions,and the transcripts of Ehd1,RFT1,and Hd3 a were distinctly reduced,while other flowering genes were not affected.SDG722 partially affected H3K4me3 and mainly affected the total abundance of H3K36me3 in rice,indicating that the methyltransferase SDG722 positively regulated heading date by promoting the deposition of H3K36me3.SDG720 also encoded a SET-domain protein,which was a homolog of Arabidopsis ATXR5.The loss of SDG720 function led to a late-flowering phenotype under long-day conditions instead of short-day conditions,demonstrating that SDG720 relied on photoperiod to modulate flowering time.Moreover,SDG720 affected the establishment of H3K4me3 and H3K36me3 in rice,indicating that the methyltransferase SDG720 promoted flowering by advancing the establishment of H3K4me3 and H3K36me3.However,overexpressing neither SDG722 or SDG720 could reverse the non-flowering of rid1,suggesting that both SDG722 and SDG720 functioned upstream of RID1 to modulate rice floral transition.6.SLR1 competes with SDG722 and Os MED25 to bind to the C-terminal protein of RID1Pull-down and quantitative split-LUC assays demonstrated that RID1 interacted with Os MED25 and SDG722.Competitive pull-down and quantitative split-LUC assays further demonstrated that SLR1 inhibited the interaction among RID1,Os MED25 and SDG722,indicating that SLR1 might condense the chromatin of Hd3 a and RFT1 by reducing the ability of RID1 recruiting epigenetic modifiers.Interestingly,SLR1 could also interact with Os MED25 and SDG722,suggesting that SLR1 played a flowering repressive role in a complicated way.7.The RID1 complex influences the chromatin status of Hd3 a and RFT1 promoter regionsMNase-q PCR results showed that the transcriptional start regions of Hd3 a and RFT1 became tight in rid1 young leaves and mature leaves.Moreover,functional loss of RID1 resulted in a remarkable reduction in the deposition of H3K9 ac,H3K4me3,and H3K36me3 at Hd3a and RFT1 loci,while H3K27me3 hardly changed.However,RID1 did not influence the total abundance of H3K9 ac,H3K4me3,and H3K36me3.Floral transition in plants is a complicated and delicate regulation process,this study establishes a molecular model of RID1 recruiting different interacting proteins to regulate rice floral transition.RID1 recruits Os MED25,SDG720 or SDG722 to form protein complexes,thereby changing the histone modification and chromatin accessibility of downstream genes Hd3 a and RFT1,while SLR1 inhibits this recruitment effect of RID1.Furthermore,we analyze the pathways of rice DELLA protein SLR1,key mediator subunit Os MED25,methyltransferases SDG720 and SDG722 that modulate rice flowering.Therefore,we consider that the recruitment of epigenetic modification proteins Os MED25,SDG720,and SDG722 by RID1 is vital for rice floral transition.